Use of the total coumarins of Cnidium monnieri in preparation of the medicament for treating psoriasis

ABSTRACT

The present invention relates to a use of total coumarins of cnidium fruit in preparation of a medicament for treating psoriasis. The invention can significantly inhibit mitosis of vaginal epithelial cells of rats; promote the formation of epidermal granular stratum in rat tail&#39;s scale; inhibit passive cutaneous anaphylaxis of rats; significantly reduce ear-swelling of mouse induced by croton oil and foot-swelling of rats induced by egg-white; and lessen the itching reaction caused by increasing itch-causing threshold of guinea pigs caused by histamine phosphate. The total coumarins of the invention can improve the clinical symptom of psoriasis, have higher curative effect and are safe to use. It can be administrated both externally and orally.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to an extract of cnidium fruit, and a usethereof, particularly total coumarins extracted from cnidium fruit aswell as a use thereof in the treatment of psoriasis.

TECHNICAL BACKGROUND OF THE INVENTION

Psoriasis is a kind of a chronic skin disorder most often positioned onthe scalp, elbows, knees, and lower back of the patient, which presentssilvery scales covering affected parts. The patient severely suffersfrom it. As a result, psoriasis significantly impacts patients'daily-life and work.

Recently a great number of patients have been suffering from psoriasis.However, psoriasis is very difficult to cure, and has been considered bythe WHO one of the most important diseases to treat and prevent in thedermatological field.

The pathogeny of psoriasis has not been very clear. It is generallyconsidered related with complicated factors such as heredity, infection,immunity, metabolizability, incretion, inflammation and psychologicalaspects.

Currently, it is the main way to treat psoriasis with medicaments thougha lot of methods have been used. However, it is too difficult to avoidrelapsing. By now cortical hormones or immunosuppressants have been usedfor the treatment (see: Progress of External Cortical HormoneMedicaments, Yaoxuetongbao, 1985, 20(9). Although the cortical hormonecan reach a good short-term effect, it will make the epidermis anddermis of the affected part thinner, causes side effects such as inducetelangiectasis and addiction of the medicaments and induces othercomplications, if used for a long time. Meanwhile, these medicamentscost expensive.

Chinese medicine cnidium fruit (also called “fructus cnidii” or “commoncnidium fruit”), used for the treatment of diseases was recorded inancient medical books. Cnidium fruit has been used in clinic fortreating surgical, gynecological, dermatological and otolaryngologicaldiseases such as lichen, scabies and scrotum eczema, because the Chinesebelieved that cnidium fruit has effect of “wetness-removing”,“worms-killing” and “itch-stopping”. However, there have been no reportson the treatment for psoriasis with total coumarins of an extract ofcnidium fruit.

SUMMARY OF THE INVENTION

An object of the present invention is to provide total coumarinsextracted from cnidium fruit, and another object of the presentinvention is to provide a use of the total coumarins defined herein inpreparing a medicament to treat psoriasis, which has curative withlittle side-effect.

In the invention, the total coumarins of cnidium fruit are extractedfrom the fruit of Cnidium monnieri (L.) Cusson. Main components of theextract are coumarin compounds, which account for, over 85% by weight onthe base of the extract. The total coumarins mentioned herein includesix compounds of coumarin, in which Osthal is of the highest content,over 90% by weight. Osthal (compound VI) is a main active compound inthe extract of cnidium fruit. All other five active compounds arefurocoumarin, which have been identified xanthotoxol (compound I),xanthotoxin (compound II), isopimpinellin (compound III), bergapten(compound IV), imperatorine (compound V).

The coumarins described in the present invention have been identifiedand separated by HPLC, and UV absorption spectrum of the six coumarinsshowed osthal: λ_(max) 322 nm (lgε |=3.9); xanthotoxol: λ_(max) 250 nm(lgε=4.25); xanthotoxin: λ_(max) 248 nm (lgε=4.4); isopimpinellin:λ_(max) 268 nm (lgε=4.26); bergapten: λ_(max) 249 nm (lgε=4.38); andimperatorin: λ_(max) 249 nm (lgε=3.65).

According to the theory that the UV absorption spectrum of allcoumarinsis can be combined to calculate the amount of the coumarins,the UV absorption spectrum of the total coumarins in ethanol has beengiven.

11 batches of extracts of cnidium fruit have been identified through UVin ethanol in the present invention and the result showed that they hadalmost the same absorption spectrum. The maximum absorption of all theextracts was at around 322 nm (from 319 nm to 326 nm) with a flat peak,and other peaks were at 250 nm, 255 nm, and 265 nm, respectively.

The present invention relates to a use of total coumarins in preparing amedicament to treat psoriasis, and particularly to a use of a Chinesetraditional medicine cnidium fruit in treating psoriasis. In theinvention, the medicament comprises 5-35% total coumarins by weight anda pharmaceutically acceptable carrier and/or excipient.

The preparation of total coumarins involved in the present invention isas follows:

Cnidium fruit was ground to be powder. The powder was extracted with80-95% ethanol at 60-80° C. for two hours, and filtered. The residuethen was extracted twice with 80-95% ethanol. The filtrates werecombined and kept at room temperature for at least 16 hours, andfiltered to obtain a green precipitate. Further precipitate was obtainedby concentrating the filtrate. The total coumarins of the invention werethen obtained after the precipitate was dried.

1. Effect of Total Coumarins on Mitosis of Vaginal Epithelial Cells inMice

60 female NIH mice of 27±2 g were selected. After beingintraperitioneally injected with diethylstilbesrolum for 3 dayscontinuously, 0.2 mg/day for each animal, all the mice were left inestrum interval. From the 4th day, the mice were randomly divided intofour groups, 15 mice each. The mice of the four groups wereadministrated for three days by gavage with 150 mg/kg of totalcoumarins, 75 mg/kg of total coumarins, 15 mg/kg of anthralin and 15mg/kg of physiological saline, respectively, once one day. The lastadministration was carried out at 8:00 am in order to avoid theinterference of various administration rhythms between day and night inthe mice to the test result. At 9:00 am, 2 mg/kg of colchicine wasinjected to the mice to make the mitosis of vaginal epithelial cellsstopped at the middle cycle of M phase.

The mice were killed at 2 p.m. A sample from the vagina of the animalwas taken out and fixed in a 10% formaldehyde solution. A slice oftissue was observed under an optical microscope. The number of mitosisper 300 fundus cells was counted to obtain an average number of mitosisamong 100 fundus cells, which was regarded as the index of mitosis ofvaginal epithelial cells.

The result of the test was given in Table 1. It showed that the totalcoumarins of the invention had significant inhibition against mitosis ofvaginal epithelial cells in mice.

TABLE 1 Doses Index of Groups (mg/kg) Mice Mitosis % Total Coumarins 15015 11.3 ± 2.0* Total Coumarins  75 15 19.8 ± 2.8** Anthralin  15 15 13.4± 2.1* Physiological Saline  15 ml 15 22.2 ± 2.4 *P < 0.01; **P < 0.05vs Physiological Saline Group2. Effect of Ointments of Total Coumarins on Formation of StratumGranulosum Epidermidis in Rats Tail Scale

40 SD rats, 180 g+10 g, were divided randomly into four groups, of whichone group was anointed with an ointment prepared by total coumarins ofthe invention onto the tail, with a dosage of 0.3 g for each rat, andonce per day; and another group was anointed with the same ointment andthe same dosage, but twice per day. The third group was anointed with anointment prepared by anthralin with the same dosage, twice per day, andthe fourth group was anointed with a blank ointment, twice per day.After the ointment was continuously anointed onto the tail for 20 days,the rats were killed.

A piece of dorsal skin of the rat about 1.5 cm far from the tail rootwas cut down. The dorsal skin was prepared as a tissue slice, which wasobserved under an optical microscope. The number of the squama that hadconsecutive granular cells of stratum among 100 squamae in the epidermisat the rat tail was obtained. The result was given in Table 2. It showedthat the ointment made of total coumarins could greatly increase thenumber of squamae that had epidermal granular cells of stratum at therat tail.

TABLE 2 Number of Squamae Having Doses Granular Layer Groups (g/rat)Rats Cells (%, x ± SD) Coumarins Ointment 0.3 10 25.36 ± 9.28* CoumarinsOintment 0.3 10 18.45 ± 8.78** Anthralin Ointment*** 0.3 10 21.76 ±8.51* Blank Ointment 0.3 10 10.87 ± 5.64 *P < 0.01; **P < 0.05; ***onceone day3. Inhibition of Passive Cutaneous Anaphylaxis (PCA) of Rats

Hair on the back of 40 SD rats, 180 g+10 g, was shaved off. 0.2 ml of adiluted rat-antiserum (1:10) was hypodermically injected into each ratthrough two places on the back, 0.1 ml for each place. After beinginjected, the rats were divided randomly into four groups, which wereadministered by gavage with 75 mg/kg of total coumarins, 150 mg/kg oftotal coumarins, 15 mg/kg of anthralin and 15 ml/kg of distilled waterfor 3 days, once per day. After the injection with the rat-antiserum 48hours, 1 ml of 0.5% evans blue, as an antigen, was intravenouslyinjected into the rats through the caudal vein. The rats were killed in20 minutes. A piece of skin with blue speckles was taken out from theback, and immersed in 5 ml solution of acetone/physiological saline(acetone: physiological saline=7:3 v/v) for 24 hs. After centrifugation,the supernatant was taken for measuring the optical density (OD) at 590nm. Inhibition rate was obtained in accordance with the followingequation. The result was given in Table 3. It showed that totalcoumarins had a significant inhibitory effect on passive cutaneousanaphylaxis (PCA) of rats.Inhibition(%)=[OD(control)−OD(drug)]×100%/OD(control)

TABLE 3 Doses Inhibition Groups (mg/kg) Animals OD (%) Total Coumarins150 15 0.029 ± 0.018* 67.42 Total Coumarins  75 15 0.045 ± 0.027** 49.44Anthralin  15 15 0.043 ± 0.25* 51.69 Physiological Saline  15 ml 150.089 ± 0.41 *P < 0.01, **P < 0.05 vs Physiological Saline Group4. Inhibition of Total Coumarins on Ear-Swelling of Mouse Induced byCroton Oil

80 NIH mice, 27±2 g, were divided randomly into four groups, which wereanointed at the auricle of both ears with 50 ul of a croton oilcomposition composed of 2% croton oil by weight, 5% water, 20% ethanoland 73% ether by weight. 0.5 hour later, the mice of four groups wererespectively anointed at the left auricle with 0.15 g and 0.3 g of anointment prepared by total coumarins, with 0.3 g of a blank ointment,and with 0.3 g of an ointment prepared by anthralin.

After 4 hours of the treatment, the mice were killed. A piece of ear indiameter of 9 mm was taken out from both two auricles of each mouse, andweighted. The difference of the weight between the two ears was regardedas the degree of ear-swelling.

The result was listed in Table 4, which showed that the ointmentprepared by the total coumarins had a significant inhibition on theswelling at the mouse ear induced by croton oil.

TABLE 4 Differences Doses between Groups (g/ear) Animals Ears (mg)Coumarins Ointment 0.3 20 1.52 ± 0.76* Coumarins Ointment 0.15 20 0.93 ±0.68** Anthralin Ointment 0.3 20 0.95 ± 0.71** Blank Ointment 0.3 200.49 ± 0.25 *P < 0.01, **P < 0.05, vs Blank Ointment Group5. Inhibitory Effect on the Swelling of Rat Toes Induced by Egg-WriteInjection

50 SD rats, 150 g±10 g, were divided randomly into five groups. Thevolume of the toes at the same side of rats among the 5 groups wasmeasured. 10% of egg white was injected into the toes, 0.2 ml each toe.After that, rats of two groups were anointed respectively with 0.15 gand 0.3 g of an ointment of total coumarins (containing 10% totalcoumarins) at each toe. Rats of other two groups were respectivelyanointed with 0.3 g of a blank ointment and 0.3 g of an ointment ofanthralin at each toe. The rest one group was treated nothing as acontrol. 0.5 hour later, the above treatment was repeated. At 1st, 2nd,4th, and 6th hour the volume of the toes were measured. The differenceof the volume was regarded as the degree of the swelling of the toes.The result was showed in Table 5.

Table 5 showed that an ointment of total coumarins could inhibit theswelling at the mouse toes (paws) induced by egg-white.

TABLE 5 Doses Degree of Ear-Swelling (ml) Groups (g/ear) Animals 1 h 2 h4 h 6 h Coumarins Ointment 0.15 10  0.401 ± 0.210*  0.386 ± 0.183* 0.371 ± 0.167*  0.354 ± 0.152* Coumarins Ointment 0.3 10  0.324 ±0.181**  0.308 ± 0.168**  0.290 ± 0.146**  0.290 ± 0.146** Anthralin 0.310  0.408 ± 0.201*  0.391 ± 0.209*  0.375 ± 0.189**  0.351 ± 0.165*Blank Ointment 0.3 10 0.584 ± 0.205 0.579 ± 0.198 0.554 ± 0.186 0.528 ±0.181 Control 10 0.612 ± 0.207 0.608 ± 0.214 0.591 ± 0.195 0.486 ± 0.104*P < 0.05, **P < 0.01 vs Control Group6. Anti-Itching Effect on Guinea Pig Induced by Histamine Phosphate

50 guinea pigs, 250 g±15 g, were divided randomly into five groups.Right toes of the animal were shaved off. Animals of two groups wereanointed respectively with 0.15 g and 0.3 g of an ointment of totalcoumarins (containing 10% total coumarins) at each toe. Animals of twogroups were respectively anointed with 0.3 g of an ointment withouttotal coumarins and 0.3 g of an ointment of anthralin at each toe. Therest group was treated nothing as a control.

The skin where the hair was shaved off was scratched with 1^(#)sand-paper next day until some extravasate exuded. The above treatmentwas then repeated. After 10 minutes, 0.01% of a histamine phosphatesolution with a successive concentration starting at 0.01%, 0.02%, 0.03%was dropped onto the scratched toe at an interval of 3 minutes (0.05 mleach time) until the guinea pig began to lap up the toe. The dosage ofthe histamine when the guinea pig lapped up the toe was regarded as athreshold of itch-causing. The result was given in Table 6, which showedthat the ointment of total coumarins could significantly increase thethreshold of itch-causing of the guinea pig induced by histaminephosphate.

TABLE 6 Doses Itching Groups (g/toe) Animals Threshold (ug) CoumarinsOintment 0.15 10 142.75 ± 120.15* Coumarins Ointment 0.3 10 206.25 ±111.60** Anthralin Ointment 0.3 10 158.50 ± 131.48* Blank Ointment 0.310  38.75 ± 20.14 Control 10  37.50 ± 25.00 *P < 0.05, **P < 0.01 vsControl Group7. Clinical Trial

Methods: Comparing effect of total coumarins on psoriasis with that ofanthralin in patients. An ointment containing 10% total coumarins wasanointed onto psoriasis at one place of a patient and an ointment ofanthralin was anointed onto psoriasis at another place of the identicalpatient (as positive control), for 30 days, one or two times one day. Noother relevant drugs were treated during the trial.

Assay Standard of Treatment: Four Degrees Applied According to theStandard on Dermatosis Prescribed by the National Ministry of Health

The result as given in Table 7 showed that the clinic curative ratereached 60% and the total effective rate reached 96.7%.

TABLE 7 Effec- Effec- Not-Ef- Totally Groups Patients Curative tive tivefective Effective Coumarins 30 18 9 2 1 96.7% Ointment Anthralin 30 1216 2 0  100% Ointment8. Acute Toxicity

Oral Experiment:

After being fasted for 16 hrs, 100 NIH mice, 19±1 g, half male and halffemale, were divided randomly into 10 groups, each of which contained 10mice. The ratio of consecutive dosage between each group was 1:0.75.Each mouse was administered by gavage with 0.8 ml for one time. Thedaily clinical sign of toxicity and the number of death were observedfor 14 days. LD50 and the available range of 95% according to the methodof Bliss were calculated. The result showed as follows: LD50(male)=463.90 mg/kg, the available range of 95% was 406.16-529.86 mg/kg;and LD50 (female)=409.85 mg/kg, the available range of 95% was357.02-470.50 mg/kg. The animals that survived the experiment were infairly good condition with normal appetite and put on weightrespectively.

External Experiment:

(1) Acute Toxicity Test on Scratched Skin of Rabbits

16 rabbits (NZR) were denuded at the dorsal back for 150 cm2. 24 hourslater, the naked skin was scratched with 1# sand paper until the tissuefluid effused. Then, the 16 rabbits were divided randomly into fourgroups (half were male). The rabbits of three groups were respectivelyanointed with an ointment containing 10%, 20%, and 30% of totalcoumarins, 3 g for each, and those of the other were anointed with anointment without total coumarins, 5 g for each. A piece of oil paper andgauze were used to cover the treated skin. The anointed materials wereremoved 24 hours later. The common condition of the animals was observedfor 7 days. The result showed that no rabbits died and the commoncondition of the rabbits including eating, activity, and bowel movementetc. was kept in the normal way.

(2) Acute Toxicity Test on Normal Skin of Rabbits

An acute toxicity test on the normal skin of rabbits was conducted inthe same way as described in Test (1). The result showed that there wasno death in all the four groups. After the treatment the animals were ingood condition and there were no obvious different changes in actionsand discharges, with normal appetite and the common condition of therabbits including eating, activity, and bowel movement etc. was kept inthe normal way.

Conclusion

The acute toxicity test on both the normal and the scratched skin of therabbits showed that no toxicity to the animals with the treatment oftotal coumarins (10%, 20% and 30%).

9. Chronic Toxicity

3 groups of rats were divided into High, Medium and Low dosal groups andwere administrated with total coumarins, 250 mg/kg, 158 mg/kg and 100mg/kg for 90 days. As a result, one group of the rats were treatednoting as a blank control. The common condition of the rats wasobserved, and the hematology and serum clinical chemistry wereevaluated. The result showed that all the animals presented as normalexcept two rabbits in the group administrated with 250 mg/kg died andother rats in this group increased in body weight slower than those inother groups.

The above experiments show the following advantages of the presentinvention.

(1) Total coumarins of cnidium fruit of the invention can significantlyinhibit mitosis of vaginal epithelial cells in mice.

(2) Total coumarins of cnidium fruit of the invention can greatlypromote the formation of epidermal granular stratum in rat tail'sscales, and has a strong inhibitory effect on passive cutaneousanaphylaxis of rats.

(3) It can reduce ear-swelling of mouse induced by croton oil as well asfoot-swelling of rats caused by egg-white.

(4) Anti-itching experiment on guinea pigs shows that the ointment ofthe invention can lessen the reaction of itching.

(5) It is confirmed that the total coumarins can stimulate keratodereduction and have effect of anti-inflammation, anti-anaphylaxis,anti-allergy and fungus-killing.

(6) Total coumarins extracted from cnidium fruit are safe to use, andhave strong pharmacological activity with multiple components. It can beadministrated both externally and orally.

(7) Total coumarins of the invention can improve the clinical symptom ofthe psoriasis patient and increase the curative rate (60%, 96.7% of thetotal efficiency).

(8) Materials used in the present invention can be got form a plenty ofsources.

Medicaments of the invention can be prepared in various formulationssuch as capsules, syrups, injections, ointments, creams, suppositories,tinctures and so on.

EMBODIMENTS TO CARRY OUT THE INVENTION

The invention will be further described with the following examples.

EXAMPLE 1

1 kg of fruit of Cnidium monnieri (L.) Cusson was powdered, and thendissolved in 6 kg of 85% ethanol. The mixture was maintained at 80° C.for 2 hours, and filtered. The residue was extracted twice with 3,000 mlof 85% ethanol at 80° C., each for 2 hours. All filtrates were combinedand kept at room temperature. Colorful precipitate was collected byfiltration. The filtrate was then concentrated at 80° C. under reducedpressure until the concentration of ethanol in the filtrate reached 45%and kept at room temperature. After 24 hours, green precipitate wascollected and dried at 60° C. to obtain an extract of total coumarins,yield 1.2%.

EXAMPLE 2

1 kg of fruit of Cnidium monnieri (L.) Cusson was powdered, and thendissolved in 5 kg of 95% ethanol. The mixture was maintained at 60° C.for 2 hours, and filtered. The residue was extracted twice with 3,000 mlof 95% ethanol at 60° C., each for 2 hours. All filtrates were combinedand kept at room temperature. Colorful precipitate was collected byfiltration. The filtrate was then concentrated at 60° C. under reducedpressure until the concentration of ethanol in the filtrate reached 45%and kept at room temperature. After 24 hours, green precipitate wascollected and dried at 80° C. to obtain an extract of total coumarins,yield 1.2%.

EXAMPLE 3

An ointment of oil in water was prepared with total coumarins obtainedin Example 1 and other components: total coumarins 100 g, Stearic acid80 g, glyceryl monostearate 100 g, white vaseline 80 g, glycerin 160 g,Tween-80 60 g, water 280 g, and glycerin and water (4:10) were added to1,000 g. The ointment prepared was even and smooth, without irritationto the skin.

EXAMPLE 4

100 g of total coumarins prepared in Example 1 was, mixed with starch,and wetted with a little amount of ethanol. The mixture was granulatedafter mixed uniformly, and dried. Then, 1,000 tablets were prepared bytableting the granules, coating and polishing the same using aconventional method after a lubricant was added. Each tablet contained0.1 g of the total coumarins.

EXAMPLE 5

300 g of total coumarins prepared in Example 2 was mixed with starch,and wetted with a little amount of ethanol. The mixture was granulatedafter mixed uniformly, and dried. Then, 1000 tablets were prepared bytableting the granules, coating and polishing the same using aconventional method after a lubricant was added. Each tablet contained0.3 g of the total coumarins.

EXAMPLE 6

250 g of total coumarins as prepared in Example 2 was ground into finepowder and sieved. The sieved powder, encapsulated. 1,000 of capsuleswere prepared, each containing 0.25 g of the total cumarins.

EXAMPLE 7

10 g of total coumarins prepared in Example 1 was dissolved in 10 ml ofbenzyl alcohol and 10 ml of Tween-80. To the mixture was added injectionwater to reach 2,000 ml. The solution was filtered, sterilized, andbottled with 2 ml each. The injection per milliliter contained 5 mg ofthe total coumarins.

EXAMPLE 8

20 g of total coumarins prepared in Example 2 was dissolved in 200 mlbenzyl alcohol and, 20 ml of propylene glycol. To the mixture was addedinjection water to reach 2,000 ml. The solution was, filtrated,sterilized, and bottled with 2 ml each. The injection per millilitercontained 10 mg of the total coumarins.

The invention claimed is:
 1. A method of treating psoriasis, comprisinga step of administering a pharmaceutically effective amount of apharmaceutical composition comprising an active component and a carrier,wherein said active component comprises a major active ingredient and agroup of minor active ingredients, said major active ingredient isosthol which accounts for at least 90% by weight of said activecomponent, and said pharmaceutical composition possesses an inhibitoryeffect on epithelial cell mitosis and a stimulating effect on formationof stratum granulosum epidermidis.
 2. The method of claim 1, whereinsaid active component accounts for 5-35% by weight of saidpharmaceutical composition which is suitable for a long term treatmentcourse of at least 90 days.
 3. The method of claim 2, wherein said groupof minor active ingredients comprises a pharmaceutically effectiveamount of each xanthotoxol, xanthotoxin, isopimpinellin, bergapten, andimperatorine.
 4. The method of claim 3, wherein said pharmaceuticalcomposition is in a dosage form of an ointment.
 5. The method of claim3, wherein said pharmaceutical composition is in a dosage form of atablet.
 6. The method of claim 3, wherein said pharmaceuticalcomposition is in a dosage form of an injection.
 7. The method of claim1, wherein said group of minor active ingredients comprises apharmaceutically effective amount of xanthotoxol.
 8. The method of claim1, wherein said group of minor active ingredients comprises apharmaceutically effective amount of xanthotoxin.
 9. The method of claim1, wherein said group of minor active ingredients comprises ofisopimpinellin.
 10. The method of claim 1, wherein said group of minoractive ingredients comprises a pharmaceutically effective amount ofbergapten.
 11. The method of claim 1, wherein said group of minor activeingredients comprises a pharmaceutically effective amount ofimperatorine.
 12. The method of claim 1, wherein said group of minoractive ingredients comprises a pharmaceutically effective amount of eachxanthotoxol, xanthotoxin, isopimpinellin, bergapten, and imperatorine.13. The method of claim 1, wherein said pharmaceutical composition is ina dosage form of an ointment.
 14. The method of claim 1, wherein saidpharmaceutical composition is in a dosage form of a tablet.
 15. Themethod of claim 1, wherein said pharmaceutical composition is in adosage form of an injection.